Abstract DESCRIPTION: It is estimated that over 500,000 bone grafting procedures are performed each year in the United States with over 50% of them being done in the setting of spine fusions, which is one of the toughest clinical bone healing environments. Orthopaedic surgical treatment of these problems typically requires bone grafting, yet iliac crest bone graft may fail to heal in 25-45% of patients. Numerous bone substitutes, electrical stimulation, and stem cells have failed to show sufficient potency to initiate bone healing. The first recombinant bone morphogenetic protein (rhBMP-2) was approved in 2002 and represented a major advance by demonstrating unprecedented bone healing potential. Unfortunately, the high dose made routine use of rhBMP-2 economically unfeasible in most bone healing situations and local side effects (swelling, hematoma, inflammation, transient bone resorption) along with the possibility of increased cancer risk have further slowed its adoption. This proposal will describe a novel approach to initiate local bone formation without the use of supra-physiologic doses of recombinant BMPs or the use of exogenous stem cells. RESEARCH PLAN: In this proposal, we provide a well-designed process for identifying and screening repurposed drugs for the clinical challenge of spine fusion. Through a computerized drug design and screening process we identified a family of small molecules which are capable of displacing a repressor (FKBP12) off the BMP-receptor, thereby enabling local activation of the bone formation cascade. Tacrolimus and rapamycin are already FDA-approved as safe local and systemic anti-inflammatory/immunosuppressant drugs, making them ideal to be repurposed as local osteogenic agents. The success of this work will provide profound evidence that an already FDA-approved immunosuppressant small molecule may be applied towards a new indication, bone regeneration for spine fusion. SPECIFIC AIMS: Aim 1: Target validation of tacrolimus- and rapamycin-osteoinduction in mesenchymal stem cells (MSCs) is via activation of the BMP receptor. Aim 2: Local delivery of tacrolimus or rapamycin induces dose-dependent in vivo bone formation. Aim 3: Tacrolimus or rapamycin can induce bone fusion in a clinically relevant, orthotopic spine fusion rabbit model. METHODS: The first aim will expand on preliminary data and validate the binding of the small molecules to the target region of the BMP receptor. Aim 2 will establish a therapeutic dose range for induction of ectopic bone by the small molecules and test the effects of a compression resistant nanofiber mesh to enhance retention at the site of delivery and improve bone formation. The third aim will test one-time local administration of the small molecules in a well-established pre-clinical model rabbit spine fusion (initiating new bone formation).